The present invention relates to novel antimicrobial peptides and methods of making and using such peptides to inhibit microbial growth and in pharmaceutical compositions for treatment or prevention of infections caused by a broad range of microorganisms including gram-positive and gram-negative bacteria, especially Pseudomonas aeruginosa and Acinetobacter baumannii. 
The extensive clinical use of classical antibiotics has led to the growing emergence of many medically relevant resistant strains of bacteria (1,2). Moreover, only three new structural classes of antibiotics (the oxazolidinone, linezolid, the streptogramins and the lipopeptide-daptomycin) have been introduced into medical practice in the past 40 years. Therefore, the development of a new class of antibiotics has great significance. The cationic antimicrobial peptides could represent such a new class of antibiotics (3-5). Although the exact mode of action of the cationic antimicrobial peptides has not been established, all cationic amphipathic peptides interact with membranes and it has been proposed that the cytoplasmic membrane is the main target of some peptides, where peptide accumulation in the membrane may cause increased permeability and loss of barrier function (6,7). Therefore, the development of resistance to these membrane active peptides is less likely because this would require substantial changes in the lipid composition of cell membranes of microorganisms.
Two major classes of the cationic antimicrobial peptides are the α-helical and the β-sheet peptides (3,4,8,9). The β-sheet class includes cyclic peptides constrained in this conformation either by intramolecular disulfide bonds, e.g., defensins (10) and protegrins (11), or by an N-terminal to C-terminal covalent bond, e.g., gramicidin S (12) and tyrocidines (13). Unlike the β-sheet peptides, α-helical peptides are more linear molecules that mainly exist as disordered structures in aqueous media and become amphipathic helices upon interaction with the hydrophobic membranes, e.g., cecropins (14), magainins (15) and melittins (16).
The major barrier to the use of antimicrobial peptides as antibiotics is their toxicity or ability to lyse eukaryotic cells, at least in some instances. This is perhaps not a surprising result if the target is indeed the cell membrane (3-6). To be useful as a broad-spectrum antibiotic, it is necessary to dissociate deleterious effects on mammalian cells from antimicrobial activity, i.e., to increase the antimicrobial activity and reduce toxicity to normal cells.
A synthetic peptide approach to examining the effect of changes, including small or incremental changes in hydrophobicity/hydrophilicity, amphipathicity and helicity of cationic antimicrobial peptides can facilitate rapid progress in rational design of peptide antibiotics. Generally, L-amino acids are the isomers found throughout natural peptides and proteins; D-amino acids are the isomeric forms rarely seen in natural peptides/proteins, except in some bacterial cell walls. In certain circumstances, the helix-destabilizing properties of D-amino acids offer a potential systematic approach to the controlled alteration of the hydrophobicity, amphipathicity, and helicity of amphipathic α-helical model peptides (26).
A particular structural framework of an amphipathic α-helical antimicrobial peptide, V681 (28) and its related peptide D1, has been used to change peptide amphipathicity, hydrophobicity, net charge and helicity by single D- or L-amino acid substitutions in the center of either the polar or nonpolar faces of the amphipathic helix so that the effects on antimicrobial activity and host toxicity can be determined. Portions of this work have been described in International Patent Publication WO 2006/065977 and U.S. Ser. No. 61/195,299, which are incorporated by reference herein. See also references 53, 92-94.
By introducing different D- or L-amino acid substitutions, it was shown that hydrophobicity, amphiphilicity and helicity have dramatic effects on the biophysical and biological activities and, thus that significant improvements in antimicrobial activity and specificity can be achieved. High peptide hydrophobicity and amphipathicity can result in greater peptide self-association in solution. Temperature profiling in reversed-phase chromatography has proven useful for measuring self-association of small amphipathic molecules (29,30). This technique has been applied to the investigation of the influence of peptide dimerization ability on biological activities of α-helical antimicrobial peptides.
Widespread bacterial resistance to all commercially available antibiotic classes and their respective mechanisms of action is well documented (102). Recent reports reveal that the incidence of resistant gram-positive and gram-negative bacteria isolates generated in hospital patients exceeds 25% in several EU Member States (103). Bacterial resistance to antibiotics is having a dramatic impact on the global healthcare system. For example, 37,000 patients die in the EU annually from a multidrug-resistant hospital-acquired infection, resulting in healthcare costs of at least EUR 1.5 billion ($2.3B) each year (103), while in the U.S., annual healthcare costs related to the treatment of P. aeruginosa, alone, is estimated at $2.7 billion (104). Despite the tremendous expenditures to treat the problem, the CDC estimates that 99,000 deaths occurred in the U.S. in 2007 due to resistant infections within the healthcare system (105).
There is a long felt need in the art for new antibiotics to circumvent the development of resistance to many of the antibiotics currently in use and for new antibiotics with relatively low toxicity for use in human and veterinary medicine, especially for use in treatments for infections with multiply drug resistant and/or difficult to treat microorganisms.